Traumatic injury to peripheral nerves results in the loss of neural functions. Recovery by regeneration depends on the cellular and molecular events of Wallerian degeneration that injury induces distal to the lesion site, the domain through which severed axons regenerate back to their target tissues. Innate-immunity is central to Wallerian degeneration since innate-immune cells, functions and molecules that are produced by immune and non-immune cells are involved. The innate-immune response helps to turn the peripheral nerve tissue into an environment that supports regeneration by removing inhibitory myelin and by upregulating neurotrophic properties. The characteristics of an efficient innate-immune response are rapid onset and conclusion, and the orchestrated interplay between Schwann cells, fibroblasts, macrophages, endothelial cells, and molecules they produce. Wallerian degeneration serves as a prelude for successful repair when these requirements are met. In contrast, functional recovery is poor when injury fails to produce the efficient innate-immune response of Wallerian degeneration.

Figure 4.The cytokine network of Wallerian degeneration.

Injury sets in motion the cytokine network of normal Wallerian degeneration. Intact myelinating Schwann cells enwrap intact axons and further express normally the inflammatory cytokines TNFα and IL-1α mRNAs and the TNFα protein. Traumatic injury at a distant site in the far left (not shown) induces the rapid upregulation of TNFα and IL-1α mRNAs expression and proteins production and secretion by Schwann cells within 5 hours. The nature of the signal(s) that are initiated at the injury site, travel down the axon, and then cross over to Schwann cells are not known (?). Concomitantly, Schwann cell derived TNFα and IL-1α induce resident fibroblasts to upregulate the expression of cytokines IL-6 and GM-CSF mRNAs and the production and secretion of their proteins within 2 to 5 hours after the injury. Inflammatory IL-1β mRNA expression and protein production and secretion are induced in Schwann cells with a delay of several hours. The expression of chemokines MCP-1/CCL2 and MIP-1α/CCL3 are upregulated by TNFα, IL-1β and IL-6 as of day 1 after the injury in Schwann cells, and possibly also in fibroblasts and endothelial cells. In turn, circulating monocytes begin their transmigration into the nerve tissue 2 to 3 days after the injury. Fibroblasts begin producing apolipoprotein-E (apo-E) and Schwann cells Galectin-3/MAC-2 (Gal-3) just before the onset of monocyte recruitment. Apolipoprotein-E and Galectin-3/MAC-2 may drive monocyte differentiation towards M2 phenotype macrophage which further produces apolipoprotein-E and Galectin-3/MAC-2. Macrophages efficiently produce IL-10 and IL-6 and much less TNFα, IL-1α, IL-1β. The anti-inflammatory cytokine IL-10, aided by IL-6, down-regulates productions of cytokines. Schwann cells and fibroblasts produce also LIF. Arrows indicate activation and broken lines down-regulation. Not all possible interactions and molecules produced are shown (e.g. autocrine interactions and the role of GM-CSF inhibitor); see text for additional information. The break-down of axons and myelin, and their phagocytosis are not illustrated here; see, however, Figure 1 and Figure 2.

Myelin down-regulates myelin phagocytosis by microglia and macrophages through interactions between CD47 on myelin and SIRPalpha (signal regulatory protein-alpha) on phagocytes

BACKGROUND:Traumatic injury to axons produces breakdown of axons and myelin at the site of the lesion and then further distal to this where Wallerian degeneration develops. The rapid removal of degenerated myelin by phagocytosis is advantageous for repair since molecules in myelin impede regeneration of severed axons. Thus, revealing mechanisms that regulate myelin phagocytosis by macrophages and microglia is important. We hypothesize that myelin regulates its own phagocytosis by simultaneous activation and down-regulation of microglial and macrophage responses. Activation follows myelin binding to receptors that mediate its phagocytosis (e.g. complement receptor-3), which has been previously studied. Down-regulation, which we test here, follows binding of myelin CD47 to the immune inhibitory receptor SIRPalpha (signal regulatory protein-alpha) on macrophages and microglia.METHODS:CD47 and SIRPalpha expression was studied by confocal immunofluorescence microscopy, and myelin phagocytosis by ELISA.RESULTS:We first document that myelin, oligodendrocytes and Schwann cells express CD47 without SIRPalpha and further confirm that microglia and macrophages express both CD47 and SIRPalpha. Thus, CD47 on myelin can bind to and subsequently activate SIRPalpha on phagocytes, a prerequisite for CD47/SIRPalpha-dependent down-regulation of CD47+/+ myelin phagocytosis by itself. We then demonstrate that phagocytosis of CD47+/+ myelin is augmented when binding between myelin CD47 and SIRPalpha on phagocytes is blocked by mAbs against CD47 and SIRPalpha, indicating that down-regulation of phagocytosis indeed depends on CD47-SIRPalpha binding. Further, phagocytosis in serum-free medium of CD47+/+ myelin is augmented after knocking down SIRPalpha levels (SIRPalpha-KD) in phagocytes by lentiviral infection with SIRPalpha-shRNA, whereas phagocytosis of myelin that lacks CD47 (CD47-/-) is not. Thus, myelin CD47 produces SIRPalpha-dependent down-regulation of CD47+/+ myelin phagocytosis in phagocytes. Unexpectedly, phagocytosis of CD47-/- myelin by SIRPalpha-KD phagocytes, which is not altered from normal when tested in serum-free medium, is augmented when serum is present. Therefore, both myelin CD47 and serum may each promote SIRPalpha-dependent down-regulation of myelin phagocytosis irrespective of the other.CONCLUSIONS:Myelin down-regulates its own phagocytosis through CD47-SIRPalpha interactions. It may further be argued that CD47 functions normally as a marker of "self" that helps protect intact myelin and myelin-forming oligodendrocytes and Schwann cells from activated microglia and macrophages. However, the very same mechanism that impedes phagocytosis may turn disadvantageous when rapid clearance of degenerated myelin is helpful.

The cytoskeleton plays a dual role of activation and inhibition of myelin and zymosan phagocytosis by microglia

Miri Gitik, Fanny Reichert and Shlomo Rotshenker. FASEBJournal,2010

Abstract

A major innate immune function of microglia in the central nervous system is receptor mediated phagocytosis of tissue debris and pathogens. We studied how phagocytosis of degenerated myelin (i.e. tissue debris) and zymosan (i.e. yeast pathogen) are regulated by the cytoskeleton through myosin light chain kinase (MLCK) and the small GTPase Rho and its effector Rho-kinase (ROCK) in primary mouse microglia. Our observations suggest a dual role of activation and inhibition of phagocytosis by MLCK and Rho/ROCK signaling. MLCK activated whereas Rho/ROCK down-regulated complement receptor-3 (CR3) mediated phagocytosis of C3bi-opsonized and non-opsonized myelin. These opposing roles of MLCK and Rho/ROCK depended on the preferential spatial localization of their distinctive functions. MLCK further activated and Rho/ROCK down-regulated phagocytosis of non-opsonized zymosan by non-opsonic receptors (e.g. Dectin-1). In contrast, MLCK down-regulated but Rho/ROCK activated CR3-mediated phagocytosis of C3bi-opsonized zymosan. Thus MLCK and Rho/ROCK can each activate or inhibit phagocytosis but always act in opposition. Whether activation or inhibition occurs depends on the nature of the phagocytosed particle (C3bi-opsonized or non-opsonized myelin or zymosan) and the identity of receptors mediating each phagocytosis.

Fig. 2. (1) Microglia phagocytose/internalize myelin and zymosan. Primary BalbC microglia were presented with either myelin for 60 minutes or zymosan for 30 minutes, unphagocytosed particles washed out, and myelin, zymosan and F-actin studied by immunofluorescence confocal microscopy. (A) Myelin is visualized by immuncytochemistry (red), F-actin by Alexa Fluor 488 labeled phalloidin (green), and overlap (yellow). (B) Zymosan is Alexa Fluor 594 labeled (red), F-actin (green), and overlap (yellow). Optical slices, 1µm thick, were scanned sequentially, and the entire phagocyte reconstructed (top left). Horizontal (bottom) and vertical (right) sections through the entire depth of the phagocyte reveal that myelin (A) and zymosan (B) are internal to and to a large extent encircled by F-actin that is associated with the phagosome. Bars in these and all forthcoming micrographs: 5µm. (C) A phagocytic cup and phagosome in zymosan phagocytosis. F-actin is visualized by Rhodamin-labeled phalloidin (red) and zymosan by Alexa Fluor 488 labeled phalloidin (green). A single optical slice that was sequentially scanned is shown.

(3) (G) Normal microglia exhibit F-actin that is localized to the periphery and further runs throughout the cytoplasm such that filaments can be identified. (H) Similar morphology is displayed by myosin light chain (MLCK)-inhibited microglia treated by MLCK-inhibitor (MLCK-in). (J) Rho-inhibited microglia treated by exoenzyme C3-transferase (C-3 toxin) exhibit an altered branched morphology and disruption of F-actin at the cell center. F-actin is visualized by Alexa Fluor 488 labeled phalloidin (green). Optical slices, 1µm thick, were scanned, and the entire phagocyte reconstructed. Shown are slices within 1 to 2µm from bottom.

(4) The phagocytic cup in myelin phagocytosis is characterized by the colocalization of myelin, F-actin and p-MLC (I, K & L). Microglia were exposed to myelin for 5 to 7 minutes, unbound/unphagocytosed myelin washed out, and myelin, F-actin and p-MLC visualized. (I) Myelin (blue) and p-MLC (red) are visualized by immunocytochemistry, F-actin by Alexa Fluor 488 labeled phalloidin (green), and overlap (yellow). The same microglia is shown again, but either myelin/F-actin (K) or myelin/p-MLC (L) are displayed. Optical slices, 1µm thick, were scanned sequentially, and the entire phagocyte reconstructed (top left). Horizontal (bottom) and vertical (right) sections through the entire depth of microglia reveal that myelin, F-actin and p-MLC are colocalized.

Dissimilar and similar functional properties of complement receptor-3 inmicroglia and macrophages in combating yeast pathogens by phagocytosis

Smadar Hadas, Fanny Reichert and Shlomo Rotshenker. GLIA, 2010

Abstract

CNS microglia and peripheral tissue macrophages remove pathogens by phagocytosis. Zymosan, a model yeast pathogen, is a β-glucan rich particle that readily activates the complement system and then becomes C3bi-opsonized. CR3 (complement receptor-3) has initially been implicated in mediating the phagocytosis of both C3bi-opsonized and non-opsonized zymosan by macrophages through C3bi and β-glucan binding sites, respectively. Later, the role of CR3 as a phagocytic β-glucan receptor has been questioned and the supremacy of β-glucan receptor Dectin-1 advocated. We compare here between primary mouse CNS microglia and peripheral tissue macrophages with respect to CR3 and Dectin-1 mediated phagocytosis of C3bi-opsonized and non-opsonized zymosan. We report that microglia and macrophages display similar as well as dissimilar functional properties in this respect. Whereas CR3 and Dectin-1 function both as β-glucan/non-opsonic receptors in microglia during non-opsonized zymosan phagocytosis, Dectin-1, but not CR3, does so in macrophages. CR3 functions also as a C3bi/opsonic receptor in microglia and macrophages during C3bi-opsonized zymosan phagocytosis, leading to phagocytosis which is more efficient than that of non-opsonized zymosan. Dectin-1 contributes, albeit less than CR3, to phagocytosis of C3bi-opsonized zymosan in microglia and further less in macrophages, suggesting that C3bi-opsonization does not block all β-glucan sites on zymosan from binding Dectin-1 on phagocytes. Thus altogether CR3 and Dectin-1 contribute both to phagocytosis of non-opsonized and C3bi-opsonized zymosan in microglia whereas macrophages switch from CR3-independent/Dectin-1-dependent phagocytosis of non-opsonized zymosan to phagocytosis of C3bi-opsonized zymosan where CR3 dominates over Dectin-1.

Fig. 1. Primary microglia and macrophages that phagocytose/internalize zymosan express Dectin-1 and complement receptor-3 (CR3). (A) Primary CNS microglia from C57BL mice were presented with non-opsonized Alexa Fluor 594 labeled zymosan (red) for 30 minutes, unphagocytosed zymosan washed away, microglia fixed, and F-actin (filamentous actin) visualized by Alexa Fluor 488 labeled phalloidin (green). Optical slices, 1µm thick, were scanned sequentially, and the entire phagocyte reconstructed thereafter (bottom left). Horizontal (top) and vertical (right) sections through the entire depth of the phagocyte reveal that the two zymosan particles are internal to and to a large extent encircled by F-actin that is associated with the phagosome; note the overlap (yellow) between zymosan and F-actin. Similar observations were made for C3bi-opsonized zymosan in microglia, and C3bi-opsonized and non-opsonized zymosan in macrophages. (B) Microglia treated with cytochalasin-D, which inhibits phagocytosis, were exposed to zymosan and processed as described above. Ten fold fewer cytochalin-D treated microglia were associated with zymosan particles compared to non-treated microglia. A reconstructed microglia (bottom right), and horizontal (top) and vertical (left) sections through the entire depth of the phagocyte reveal that the zymosan particle is situated within a cup-like structure, is not engulfed completely, and thus not phagocytosed/internalized. Microglia (C & D) and macrophages (E & F) express Dectin-1 and CR3. Dectin-1 and CR3 are visualized (red) by immunofluorescence confocal microscopy using anti-Dectin1 mAb 2A11 and anti-CR3 mAbs M1/70 and 5C6 combined. All bars are 5µm.